2002 Fiscal Year Final Research Report Summary
Topological mapping of unknown proteins using fluorescent probes
| Project/Area Number |
12460150
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied molecular and cellular biology
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| Research Institution | OSAKA UNIVERSITY |
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Principal Investigator |
FUKUI Kiichi Osaka University, Graduate School of Engineering, Professor, 大学院・工学研究科, 教授 (00311770)
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| Project Period (FY) |
2000 – 2002
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| Keywords | chromosome / GFP / protein / nucleus |
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| Research Abstract |
The cDNA:GFP fusion method was applied for Arabidopsis thaliana. Vector having cDNA:GFP insertion was constructed using Arabidopsis thaliana cDNA library. Screening of the transfected plants were performed according to the localizations of GFP fused proteins. As a results, a novel nuclear localized protein, At-AHM1 (Arabidopsis thaliana AT-hook motif and transmembrane domain containing protein 1), was obtained. Blast search demonstrate that this protein is unique for plents, although its homologues were found in plants. Spatial localization of At-AHM1 during the cell cycle was further investigated by using deconvolution system and it is found that in the interphase At-AHM1 localize at nuclear body with no significant co-localization with DNA. On the other hand, At-AHM1 1 migrates to the position where chromosome is present during the mitotic stage. Overexpression system of At-AHM1 using Escherichia coli was constructed. The purified recombinant protein was applied for DNA binding assay, resulting in the confirmation of DNA binding ability in vitro. Trancated At-AHM1:GFP lacking At-hook motif or transmembrane region were expressed transiently in tobacco cell line and the change in the localization pattern was observed. Localization pattern of the former mutant was similar to wild-type one, whereas the latter mutant distributed in the entire region of the cell. These results suggest that the function of At-AHM1 is fixation of DNA inside the nucleus through its transmembrane region. Also, the present research demonstrates that random cDNA:GFP fusion method is powerful technique for the screening of proteins based on the visualization technique.
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[Publications] Sone, T., Iwano, M., Kobayashi, S., Ishihara, T., Hori, N., Takata, H., Ushiki, T., Uchiyama, S., Fukui, K.: "Changes in chromosomal surface structure by different isolation conditions"Arch. Histol. Cytol. 65(5). 111-121 (2002)
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[Publications] Lee, J. H., Arumuganathan, K., Kaeppler, S. M., Park, S. H., Kim, K. Y., Chung, Y. S., Kim, D. H., Fukui, K.: "Variability of chromosomal DNA contents in maize (Zea mays L) inbred and hybrid lines"Planta. 215. 666-671 (2002)
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[Publications] Tanaka, S., Fujiwara, S., Tanaka. H., Taniguchi, M., Tabata, H., Fukui, K., Kawai, T.: "Synthesis of long poly(dA)-poly(dT) DNA without structural defects using enzymatic reaction"Chem. Comm:. 2330-2331 (2002)
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「研究成果報告書概要(欧文)」より
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